Human Stefin B Role in Cell's Response to Misfolded Proteins and Autophagy

Abstract

Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.

The small heat shock protein, HSP30, is associated with aggresome-like inclusion bodies in proteasomal inhibitor-, arsenite-, and cadmium-treated Xenopus kidney cells

“…Compared to MG132-, sodium arsenite-, or cadmium chloride-treated cells, a relatively lower number of ProteoStat dyestained structures were detected in control A6 cells. Previous studies have shown a similar phenomenon with prostate cancer, myeloma, astrocyte, and B lymphocyte cells suggesting the presence of ProteoStat-stained aggregated protein in non-stressed cells (Malek et al, 2014;Pacheco et al, 2014;Polajnar et al, 2014;Jagannathan et al, 2015;Koukourakis et al, 2015). It is possible that these control aggregates are associated with vimentin structures given their colocalization with ProteoStat dye-stained protein aggregates.…”

Proteolysis mediated by cysteine cathepsins and legumain—recent advances and cell biological challenges

“…Would a localized autophagic response be evoked in close proximity to bursting lysosomes in an attempt to rescue cells from the mishap (Meschini et al 2011;Polajnar et al 2014)? Considering that both cysteine cathepsins and legumain are involved in autophagy (Overbye et al 2011), it is conceivable they might themselves even contribute to such pathways helping clean up the cytotoxic mess they themselves have created.…”

Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation

“…Stefin B (also termed cystatin B) gene mutations, either dodecamer repeats resulting in lower protein production or missense mutations leading to misfolding, cause a progressive myoclonus epilepsy of type 1 (EPM1) with slow signs of neurodegeneration [28,29]. Similarly to cystatin C, stefin B protects neurons from excessive oxidative stress [30,31] and protein misfolding [32]. Alternative functions, such as amateur chaperone function, have also been suggested from both experimental data and bioinformatic analysis [33].…”